Vertebrate Genome Evolution: Zebrafish hox Clusters by Gaelin Kopec-Belliveau
hox Clusters and Zebrafish A hox gene specifies what a cell will grow into. Clusters of these genes are present in the anterior-posterior axis of animal embryos. They dictate the growth patterns of organisms, like where limbs and various other body parts will be places. Simple organisms like invertebrates possess one hox cluster while more complex creatures such as mammals contain four. Oddly enough however, a study conducted on zebrafish shows that they have seven hox clusters. Through gene mapping and phylogenetic analysis, researchers formed the hypothesis that there was a chromosome doubling event, most likely through whole genome duplication, that occurred sometime after the separation of lobe-finned and ray-finned fishes but before the teleost radiation that gave rise to these extra clusters. Teleosts are the one of the largest and most diverse genera of fishes, containing roughly 29,000 species and including almost half of all vertebrates. Teleosts have been shown to possess more copies of hox clusters than mammals, in spite of their much more simple anterior-posterior axis. 1 The genes in hox clusters code for DNA binding proteins that specify the patters of growth along the axes of bilateral animals. The order of these gene clusters directs the order in which they act along the body. Simple animals like invertebrate chordates have only one hox cluster while more complex organisms like tetrapods have four clusters. A group that goes against this trend is the teleost fishes. To further investigate this phenomenon researchers isolated and analyzed hox ''clusters from the zebrafish ''Dania rerio, ''a teleost.1 To begin the survey, genomic DNAs were identified in P1 artificial chromosomes (PACs). This was done using degenerate primers to amplify homeoboxes. Homeoboxes are DNA sequences found within genes that are involved in the regulatory processes of anatomical development of organisms. Overlapping PAC's were then identified and their ''hox ''gene content was analyzed using sequenced gene coding regions, redundant primers and analyzed gene phylogenies. Though this, seven ''hox clusters were identified. These clusters were found to include 40 of the 41 known zebrafish hox genes, seven previously unknown hox genes, one hox ''pseudogene and the ''evx gene. A pseudogene is a dysfunctional relation to a real gene that has lost its proton coding ability and has no function in the cell any longer.1 Phylogenetic analysis of the identified sequences grouped zebrafish genes into one of 13 homologous groups. Throughs this, a comparison could be made between gene grouping in different species. Two groups, 4 and 9, appear in every mammalian hox cluster and in four zebrafish clusters. Due to this similarity, researchers joined the nucleotide sequences of these groups and constructed a phylogenetic tree. From this they were able to show that each of the four clusters in zebrafish is orthologous to one of the four mammalian hox clusters. This indicates that the duplication event that the four mammalian clusters are the product of happened before the divergence of ray-finned and lobe-finned fish lines. An event that occurred roughly 420 million years ago. 1 Though more analysis of the sequences, the origins of the other three zebrafish hox clusters was discovered. The results of the group 6 phylogenetic tree showed that zebrafish contain two orthologs of the mammalian cluster HOXB6, these are called hoxb6a and hoxb6b. Additionally, zebrafish contain two orthologs of the mammalian cluster HOXC6 dubbed hoxc6a and hoxc6b. Zebrafish also contain two orthologs to the mammalian cluster HOXA. These findings indicate that all hox genes were copied in the line that led to zebrafish, after it split from the line that led to tetrapods.1 Comparative analysis of the clusters provided a theoretical history of HOX cluster duplication. The most supported theory is called the (AB)(CD) model and proposes two sequential duplications, producing a proto-''AB cluster and a proto-''CD ''cluster, after the first occurrence. It is thought that a duplication, most likely in an agnathan fish gave rise to a proto-''AB cluster missing group 12 and a proto-''CD'' cluster lacking groups 2 and 7. The second duplication is thought to have occurred in an ancient gnathostome fish. This caused losses of group 8 from the HOXA cluster, EVX from the HOXC cluster, group 11 from the HOXB cluster and groups 5 and 6 from the HOXD cluster. Due to these duplications and losses, the tetrapod lineages lost an EVX gene from the HOXB cluster and HOXC1 ''and ''HOXC3. Further duplications and losses have occurred since the first divergence of ray-finned and lobe-finned fish, stretching the gap between species. The study concluded that the complexity of teleosts hox ''clusters surpassed that of mammals millions of years ago. This conclusion has called into question the long regarded theory that the number of ''hox clusters an organism posses dictates its complexity. References 1Angel Amores, A. F., Yi-Lin Yan, Lucille Joly, Chris Amemiya, Andreas Fritz, Robert K. Ho, James Langeland, Victoria Prince, Yan-Ling Wang, Monte Westerfield, Marc Ekker, John H. Postlethwait. (1998). Zebrafish hox Clusters and Vertebrate Genome Evolution. Science 282(5394), 1711-1714. 2 http://www.bio.umass.edu/biology/karlstrom/FishFacility.html